HTRF-based assay for detection of mono-ADP-ribosyl hydrolyzing macrodomains and inhibitor screening

Summary The COVID-19 pandemic has highlighted the lack of effective, ready-to-use antivirals for the treatment of viruses with pandemic potential. The development of a diverse drug portfolio is therefore crucial for pandemic preparedness. Viral macrodomains are attractive therapeutic targets as they are suggested to play an important role in evading the innate host immune response, making them critical for viral pathogenesis. Macrodomains function as erasers of mono-ADP-ribosylation (deMARylation), a post-translational modification that is involved in interferon signaling. Herein, we report the development of a modular HTRF-based assay, that can be used to screen for inhibitors of various viral and human macrodomains. We characterized the five most promising small molecule SARS-CoV-2 Mac1 inhibitors recently reported in the literature for potency and selectivity and conducted a pilot screen demonstrating HTS suitability. The ability to directly detect enzymatic activity makes the DeMAR assay a valuable addition to the existing tools for macrodomain drug discovery.


SUPPLEMENTAL INFORMATION
The readout was performed with 200 nM sGFP-PARP14 Mac2/3 as described in Methods.The term "Avi-" implies that the respective macrodomain features an N-terminal biotinylated Avi-tag.The G131V mutant is sterically inactive, while the N41A mutant is catalytically inactive.However, in the N41A mutant the ADPr binding pocket is not sterically blocked, and hence, can still accommodate ADPr or bind to MARylated substrate.Consequently, the N41A mutant competes with sGFP-PARP14 Mac2/3 for binding to Tb-PARP10cat, which explains the reduction in HTRF observed with this Mac1 mutant.A DMSO control (without Mac1) is the lowest concentration in each data set.Data represent the means ± SD of 3 technical replicates each concentration tested.

Figure S4: Evaluation of the influence of different incubation times on the deMARylation of SARS-CoV-2 Mac1 in the one-pot and in the wash setup, related to Table 1.
(A) SARS-CoV-2 Mac1 was titrated from 0.08 up to 640 nM against 3 nM Tb-PARP10cat in the one-pot setup.The readout was performed with 200 nM sGFP-PARP14 Mac2/3 after 0.5, 1, 2, 3, 4, 7, 18, 22 h as described in Methods.A negative control is the lowest concentration in each data set.Data represent the means ± SD of 3 technical replicates for each point of time.(B) SARS-CoV-2 Mac1 was titrated from 0.08 up to 640 nM against 3 nM Tb-PARP10cat in the wash setup.The readout was performed with 200 nM sGFP-PARP14 Mac2/3 after 0.5, 1, 2 h as described in Methods.A negative control is the lowest concentration in each data set.Data represent the means ± SD of 3 technical replicates for each point of time.

Figure S4 :
Figure S4: Evaluation of the influence of different incubation times on the deMARylation of

Figure S5 :
Figure S5: DeMAR assay (one-pot setup) dose-response experiments of the eight hits from

Figure S6 :
Figure S6: DeMAR assay (wash setup) dose-response experiments of the eight hits from the

Figure S7 :
Figure S7: SDS-PAGE analysis of all of the protein preparations that were used for the

Figure S2 :
Figure S2: Evaluation of the influence of different glycerol and DMSO concentrations on the activity of SARS-CoV-2 Mac1, related to Figure 1.(A) SARS-CoV-2 Mac1 was titrated from 0.04 up to 640 nM against 3 nM Tb-PARP10cat.The readout was performed with 200 nM sGFP-PARP14 Mac2/3 as described in Methods.This experiment was conducted in HRTF buffer containing either 1, 2, 5 or 10 % glycerol.A DMSO control (no Mac1) is the lowest concentration in each data set.Data represent the means ± SD of 3 technical replicates for each buffer condition.(B) SARS-CoV-2 Mac1 was titrated from 0.0 up to 267 nM against 6 nM Tb-PARP10cat.The readout was performed with 200 nM sGFP-PARP14 Mac2/3 as described in Methods.This experiment was conducted in HRTF buffer containing either 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2 or 3 % DMSO.A DMSO control (no Mac1) is the lowest concentration in each data set.Data represent the means ± SD of 3 technical replicates for each buffer condition.

Figure S3 :
Figure S3: Comparison of the activity of SARS-CoV-2 Mac1 with two inactive mutants, related to Figure 1.SARS-CoV-2 Mac1 homologs were titrated from 0.08 nM up to 1.28 µM against 3 nM Tb-PARP10cat.The readout was performed with 200 nM sGFP-PARP14 Mac2/3 as described in Methods.The term "Avi-" implies that the respective macrodomain features an N-terminal biotinylated Avi-tag.The G131V mutant is sterically inactive, while the N41A mutant is catalytically inactive.However, in the N41A mutant the ADPr binding pocket is not sterically blocked, and hence, can still accommodate ADPr or bind to MARylated substrate.Consequently, the N41A mutant competes with sGFP-PARP14 Mac2/3 for binding to Tb-PARP10cat, which explains the reduction in HTRF observed with this Mac1 mutant.A DMSO control (without Mac1) is the lowest concentration in each data set.Data represent the means ± SD of 3 technical replicates each concentration tested.

Figure S5 :
Figure S5: DeMAR assay (one-pot setup) dose-response experiments of the eight hits from the Prestwick SARS-CoV-2 Mac1 pilot screen, related to Figure 5. (A) Each compound was titrated from 41 nM up to 667 µM added to 3 nM Tb-PARP10cat, and 80 nM SARS-CoV-2 Mac1 in the one-pot setup.The readout was performed with 200 nM sGFP-PARP14 Mac2/3 as described in Methods.A counter screen without Mac1 addition was also carried out for each compound titration (red).A DMSO control for each titration was performed.Data represent the means ± SD of 3 technical replicates for each concentration tested.Prestw-1472 is hexachlorophene (IC50: 114 µM).

Figure S6 :
Figure S6: DeMAR assay (wash setup) dose-response experiments of the eight hits from the Prestwick SARS-CoV-2 Mac1 pilot screen, related to Figure 5.Each compound was titrated from 82 nM up to 667 µM added to 6 nM Tb-ARTD10cat, and 80 nM SARS-CoV-2 Mac1 in the wash setup.The readout was performed with 200 nM sGFP-ARTD8 Mac2/3 as described in Methods.A DMSO control with and without Mac1 (3 technical replicates each) was performed to define the upper and lower assay plateau.Data represent the means ± SD of 3 technical replicates for each concentration tested.Prestw-1472 is hexachlorophene.

Figure S7 :
Figure S7: SDS-PAGE analysis of all of the protein preparations that were used for the DeMAR assay, related to Figure 1.For SDS-PAGE, all protein samples were adjusted to a concentration of 0.05 mg/ml.Proteins were stained with Coomassie (Instant Blue™).(A) shows all PARP enzymes used in the study.(B) shows all Mac1 homologs used in the study.